Engineering of strong, pliable tissues

ABSTRACT

It has been discovered that improved yields of engineered tissue following implantation, and engineered tissue having enhanced mechanical strength and flexibility or pliability, can be obtained by implantation, preferably subcutaneously, of a fibrous polymeric matrix for a period of time sufficient to obtain ingrowth of fibrous tissue and/or blood vessels, which is the removed for subsequent implantation at the site where the implant is desired. The matrix is optionally seeded prior to the first implantation, after ingrowth of the fibrous tissue, or at the time of reimplantation. The time required for fibrous ingrowth typically ranges from days to weeks. The method is particularly useful in making valves and tubular structures, especially heart valves and blood vessels.

RELATED APPLICATIONS

This is a divisional of U.S. Ser. No. 08/445,280 filed May 19, 1995 nowU.S. Pat. No. 5,855,610.

BACKGROUND OF THE INVENTION

This invention is generally in the field of reconstruction andaugmentation of flexible, strong connective tissue such as arteries andheart valves.

Tissue engineering is a multidisciplinary science that utilizes basicprinciples from the life sciences and engineering sciences to createcellular constructs for transplantation. The first attempts to culturecells on a matrix for use as artificial skin, which requires formationof a thin three dimensional structure, were described by Yannas and Bell(See, for example, U.S. Pat. Nos. 4,060,081, 4,485,097 and 4,458,678).They used collagen type structures which were seeded with cells, thenplaced over the denuded area. A problem with the use of the collagenmatrices was that the rate of degradation is not well controlled.Another problem was that cells implanted into the interior of thickpieces of the collagen matrix failed to survive.

U.S. Pat. No. 4,520,821 to Schmidt describes the use of syntheticpolymeric meshes to form linings to repair defects in the urinary tract.Epithelial cells were implanted onto the synthetic matrices, whichformed a new tubular lining as the matrix degraded. The matrix served atwo fold purpose—to retain liquid while the cells replicated, and tohold and guide the cells as they replicated.

In European Patent Application No. 88900726.6 “Chimeric Neomorphogenesisof Organs by Controlled Cellular Implantation Using Artificial Matrices”by Children's Hospital Center Corporation and Massachusetts Institute ofTechnology, a method of culturing dissociated cells on biocompatible,biodegradable matrices for subsequent implantation into the body wasdescribed. This method was designed to overcome a major problem withprevious attempts to culture cells to form three dimensional structureshaving a diameter of greater than that of skin. Vacanti and Langerrecognized that there was a need to have two elements in any matrix usedto form organs: adequate structure and surface area to implant a largevolume of cells into the body to replace lost function and a matrixformed in a way that allowed adequate diffusion of gases and nutrientsthroughout the matrix as the cells attached and grew to maintainviability in the absence of vascularization. Once implanted andvascularized, the porosity required for diffusion of the nutrients andgases was no longer critical.

To overcome some of the limitations inherent in the design of the porousstructures which support cell growth throughout the matrix solely bydiffusion, WO 93/08850 “Prevascularized Polymeric Implants for OrganTransplantation” by Massachusetts Institute of Technology and Children'sMedical Center Corporation disclosed implantation of relatively rigid,non-compressible porous matrices which are allowed to becomevascularized, then seeded with cells. It was difficult to control theextent of ingrowth of fibrous tissue, however, and to obtain uniformdistribution of cells throughout the matrix when they were subsequentlyinjected into the matrix.

Many tissues have now been engineered using these methods, includingconnective tissue such as bone and cartilage, as well as soft tissuesuch as hepatocytes, intestine, endothelium, and specific structures,such as ureters. There remains a need to improve the characteristicmechanical and physical properties of the resulting tissues, which insome cases does not possess the requisite strength and pliability toperform its necessary function in vivo. Examples of particularstructures include heart valves and blood vessels.

Despite major advances in its treatment over the past thirty-five years,valvular heart disease is still a major cause of morbidity and mortalityin the United States. Each year 10,000 Americans die as a direct resultof this problem. Valve replacement is the state-of-the art therapy forend-stage valve disease. Heart valve replacement with either nonlivingxenografts or mechanical protheses is an effective therapy for valvularheart disease. However, both types of heart valve replacements havelimitations, including finite durability, foreign body reaction orrejection and the inability of the non-living structures to grow, repairand remodel, as well as the necessity of life-long anticoagulation forthe mechanical prothesis. The construction of a tissue engineered livingheart valve could eliminate these problems.

Atherosclerosis and cardiovascular disease are also major causes ofmorbidity and mortality. More than 925,000 Americans died from heart andblood vessels disease in 1992, and an estimated 468,000 coronary arterybypass surgeries were performed on 393,000 patients. This does notinclude bypass procedures for peripheral vascular disease. Currently,internal mammary and saphenous vein grafts are the most frequently usednative grafts for coronary bypass surgery. However, with triple andquadruple bypasses and often the need for repeat bypass procedures,sufficient native vein grafts can be a problem. Surgeons must frequentlylook for vessels other than the internal mammary and saphenous vessels.While large diameter (0.5 mm internal diameter) vascular grafts ofdacron or polytetraflorethylene (PTFE) have been successful, smallcaliber synthetic vascular grafts frequently do not remain patent overtime. Tissue engineered blood vessels may offer a substitute for smallcaliber vessels for bypass surgery and replacement of diseased vessels.

It is therefore an object of the present invention to provide a methodfor making tissue engineered constructs which have improved mechanicalstrength and flexibility.

It is a further object of the present invention to provide a method andmaterials for making valves and vessels which can withstand repeatedstress and strain.

It is another object of the present invention to provide a methodimproving yields of engineered tissues following implantation.

SUMMARY OF THE INVENTION

It has been discovered that improved yields of engineered tissuefollowing implantation, and engineered tissue having enhanced mechanicalstrength and flexibility or pliability, can be obtained by implantation,preferably subcutaneously, of a fibrous polymeric matrix for a period oftime sufficient to obtain ingrowth of fibrous tissue and/or bloodvessels, which is then removed for subsequent implantation at the sitewhere the implant is desired. The matrix is optionally seeded prior tothe first implantation, after ingrowth of the fibrous tissue, or at thetime of reimplantation. The time required for fibrous ingrowth typicallyranges from days to weeks. The method is particularly useful in makingvalves and tubular structures, especially heart valves and bloodvessels.

Examples demonstrate construction of blood vessels, heart valves andbone and cartilage composite structures.

DETAILED DESCRIPTION OF THE INVENTION

As described herein, structures are created by seeding of fibrous orporous polymeric matrices with dissociated cells which are useful for avariety of applications, ranging from soft tissues formed of parenchymalcells such as hepatocytes, to tissues having structural elements such asheart valves and blood vessels, to cartilage and bone. In a particularimprovement over the prior art methods, the polymeric matrices areimplanted into a human or animal to allow ingrowth of fibroblastictissue, then implanted at the site where the structure is needed, eitheralone or seeded with defmed cell populations.

I. Matrix Fabrication

The synthetic matrix serves several purposes. It functions as a celldelivery system that enables the organized transplantation of largenumbers of cells into the body. The matrix acts as a scaffold providingthree-dimensional space for cell growth. The matrix functions as atemplate providing structural cues for tissue development. In the caseof tissues have specific requirements for structure and mechanicalstrength, the polymer temporarily provides the biomechanical propertiesof the final construct, giving the cells time to lay down their ownextracellular matrix which ultimately is responsible for thebiomechanical profile of the construct. The scaffold also determines thelimits of tissue growth and thereby determines the ultimate shape oftissue engineered construct. Cells implanted on a matrix proliferateonly to the edges of the matrix; not beyond.

Matrix Architecture

As previously described, for a tissue to be constructed, successfullyimplanted, and function, the matrices must have sufficient surface areaand exposure to nutrients such that cellular growth and differentiationcan occur prior to the ingrowth of blood vessels following implantation.This is not a limiting feature where the matrix is implanted andingrowth of tissue from the body occurs, prior to seeding of the matrixwith dissociated cells.

The organization of the tissue may be regulated by the microstructure ofthe matrix. Specific pore sizes and structures may be utilized tocontrol the pattern and extent of fibrovascular tissue ingrowth from thehost, as well as the organization of the implanted cells. The surfacegeometry and chemistry of the matrix may be regulated to control theadhesion, organization, and function of implanted cells or host cells.

In the preferred embodiment, the matrix is formed of polymers having afibrous structure which has sufficient interstitial spacing to allow forfree diffusion of nutrients and gases to cells attached to the matrixsurface. This spacing is typically in the range of 100 to 300 microns,although closer spacings can be used if the matrix is implanted, bloodvessels allowed to infiltrate the matrix, then the cells are seeded intothe matrix. As used herein, “fibrous” includes one or more fibers thatis entwined with itself, multiple fibers in a woven or non-woven mesh,and sponge like devices.

The matrix should be a pliable, non-toxic, injectable porous templatefor vascular ingrowth. The pores should allow vascular ingrowth and theinjection of cells in a desired density and region(s) of the matrixwithout damage to the cells. These are generally interconnected pores inthe range of between approximately 100 and 300 microns. The matrixshould be shaped to maximize surface area, to allow adequate diffusionof nutrients and growth factors to the cells and to allow the ingrowthof new blood vessels and connective tissue.

The overall, or external, matrix configuration is dependent on thetissue which is to reconstructed or augmented. The shape can also beobtained using struts, as described below, to impart resistance tomechanical forces and thereby yield the desired shape. Examples includeheart valve “leaflets” and tubes.

Polymers

The term “bioerodible”, or “biodegradable”, as used herein refers tomaterials which are enzymatically or chemically degraded in vivo intosimpler chemical species. Either natural or synthetic polymers can beused to form the matrix, although synthetic biodegradable polymers arepreferred for reproducibility and controlled release kinetics. Syntheticpolymers that can be used include bioerodible polymers such aspoly(lactide) (PLA), poly(glycolic acid) (PGA),poly(lactide-co-glycolide) (PLGA), poly(caprolactone), polycarbonates,polyamides, polyanhydrides, polyamino acids, polyortho esters,polyacetals, polycyanoacrylates and degradable polyurethanes, andnon-erodible polymers such as polyacrylates, ethylene-vinyl acetatepolymers and other acyl substituted cellulose acetates and derivativesthereof, non-erodible polyurethanes, polystyrenes, polyvinyl chloride,polyvinyl fluoride, poly(vinyl imidazole), chlorosulphonatedpolyolifins, polyethylene oxide, polyvinyl alcohol, teflon®, and nylon.Although non-degradable materials can be used to form the matrix or aportion of the matrix, they are not preferred. The preferrednon-degradable material for implantation of a matrix which isprevascularized prior to implantation of dissociated cells is apolyvinyl alcohol sponge, or alkylation, and acylation derivativesthereof, including esters. A non-absorbable polyvinyl alcohol sponge isavailable commercially as Ivalon™, from Unipoint Industries. Methods formaking this material are described in U.S. Pat. Nos. 2,609,347 toWilson; 2,653,917 to Hammon, 2,659,935 to Hammon, 2,664,366 to Wilson,2,664,367 to Wilson, and 2,846,407 to Wilson, the teachings of which areincorporated by reference herein. These materials are all commerciallyavailable.

Examples of natural polymers include proteins such as albumin, collagen,synthetic polyamino acids, and prolamines, and polysaccharides such asalginate, heparin, and other naturally occurring biodegradable polymersof sugar units. These are not preferred because of difficulty withquality control and lack of reproducible, defined degradationcharacteristics.

PLA, PGA and PLA/PGA copolymers are particularly useful for forming thebiodegradable matrices. These are synthetic, biodegradable α-hydroxyacids with a long history of medical use. PLA polymers are usuallyprepared from the cyclic esters of lactic acids. Both L(+) and D(−)forms of lactic acid can be used to prepare the PLA polymers, as well asthe optically inactive DL-lactic acid mixture of D(−) and L(+) lacticacids. Methods of preparing polylactides are well documented in thepatent literature. The following U.S. Patents, the teachings of whichare hereby incorporated by reference, describe in detail suitablepolylactides, their properties and their preparation: 1,995,970 toDorough; 2,703,316 to Schneider; 2,758,987 to Salzberg; 2,951,828 toZeile; 2,676,945 to Higgins; and 2,683,136; 3,531,561 to Trehu.

PGA is the homopolymer of glycolic acid (hydroxyacetic acid). In theconversion of glycolic acid to poly(glycolic acid), glycolic acid isinitially reacted with itself to form the cyclic ester glycolide, whichin the presence of heat and a catalyst is converted to a high molecularweight linear-chain polymer. PGA polymers and their properties aredescribed in more detail in Cyanamid Research Develops World's FirstSynthetic Absorbable Suture”, Chemistry and Industry, 905 (1970).

The erosion of the matrix is related to the molecular weights of thepolymer, for example, PLA, PGA or PLA/PGA. The higher molecular weights,weight average molecular weights of 90,000 or higher, result in polymermatrices which retain their structural integrity for longer periods oftime; while lower molecular weights, weight average molecular weights of30,000 or less, result in both slower release and shorter matrix lives.A preferred material is poly(lactide-co-glycolide) (50:50), whichdegrades in about six weeks following implantation (between one and twomonths) and poly(glycolic acid).

All polymers for use in the matrix must meet the mechanical andbiochemical parameters necessary to provide adequate support for thecells with subsequent growth and proliferation. The polymers can becharacterized with respect to mechanical properties such as tensilestrength using an Instron tester, for polymer molecular weight by gelpermeation chromatography (GPC), glass transition temperature bydifferential scanning calorimetry (DSC) and bond structure by infrared(IR) spectroscopy, with respect to toxicology by initial screening testsinvolving Ames assays and in vitro teratogenicity assays, andimplantation studies in animals for immunogenicity, inflammation,release and degradation studies.

Polymer Coatings

In some embodiments, attachment of the cells to the polymer is enhancedby coating the polymers with compounds such as basement membranecomponents, agar, agarose, gelatin, gum arabic, collagens types I, II,III, IV, and V, fibronectin, laminin, glycosaminoglycans, polyvinylalcohol, mixtures thereof, and other hydrophilic and peptide attachmentmaterials known to those skilled in the art of cell culture. A preferredmaterial for coating the polymeric matrix is polyvinyl alcohol orcollagen.

Struts

In some embodiments it may be desirable to create additional structureusing devices provided for support, referred to herein as “struts”.These can be biodegradable or non-degradable polymers which are insertedto form a more defined shape than is obtained using the cell-matrices.An analogy can be made to a corset, with the struts acting as “stays” topush the surrounding tissue and skin up and away from the implantedcells. In a preferred embodiment, the struts are implanted prior to orat the time of implantation of the cell-matrix structure. The struts areformed of a polymeric material of the same type as can be used to formthe matrix, as listed above, having sufficient strength to resist thenecessary mechanical forces.

Additives to Polymer Matrices

In some embodiments it may be desirable to add bioactive molecules tothe cells. A variety of bioactive molecules can be delivered using thematrices described herein. These are referred to generically herein as“factors” or “bioactive factors”.

In the preferred embodiment, the bioactive factors are growth factors,angiogenic factors, compounds selectively inhibiting ingrowth offibroblast tissue such as antiinflammatories, and compounds selectivelyinhibiting growth and proliferation of transformed (cancerous) cells.These factors may be utilized to control the growth and function ofimplanted cells, the ingrowth of blood vessels into the forming tissue,and/or the deposition and organization of fibrous tissue around theimplant.

Examples of growth factors include heparin binding growth factor (hbgf),transforming growth factor alpha or beta (TGFβ), alpha fibroblasticgrowth factor (FGF), epidermal growth factor (TGF), vascular endotheliumgrowth factor (VEGF), some of which are also angiogenic factors. Otherfactors include hormones such as insulin, glucagon, and estrogen. Insome embodiments it may be desirable to incorporate factors such asnerve growth factor (NGF) or muscle morphogenic factor (MMP).

Steroidal antiinflammatories can be used to decrease inflammation to theimplanted matrix, thereby decreasing the amount of fibroblast tissuegrowing into the matrix.

These factors are known to those skilled in the art and are availablecommercially or described in the literature. In vivo dosages arecalculated based on in vitro release studies in cell culture; aneffective dosage is that dosage which increases cell proliferation orsurvival as compared with controls, as described in more detail in thefollowing examples. Preferably, the bioactive factors are incorporatedto between one and 30% by weight, although the factors can beincorporated to a weight percentage between 0.01 and 95 weightpercentage.

Bioactive molecules can be incorporated into the matrix and releasedover time by diffusion and/or degradation of the matrix, they can besuspended with the cell suspension, they can be incorporated intomicrospheres which are suspended with the cells or attached to orincorporated within the matrix, or some combination thereof.Microspheres would typically be formed of materials similar to thoseforming the matrix, selected for their release properties rather thanstructural properties. Release properties can also be determined by thesize and physical characteristics of the microspheres.

II. Cells to Be Implanted

Cells to be implanted are dissociated using standard techniques such asdigestion with a collagenase, trypsin or other protease solution.Preferred cell types are mesenchymal cells, especially smooth orskeletal muscle cells, myocytes (muscle stem cells), fibroblasts,chondrocytes, adipocytes, fibromyoblasts, and ectodermal cells,including ductile and skin cells, hepatocytes, Islet cells, cellspresent in the intestine, and other parenchymal cells, osteoblasts andother cells forming bone or cartilage. In some cases it may also bedesirable to include nerve cells. Cells can be normal or geneticallyengineered to provide additional or normal function. Methods forgenetically engineering cells with retroviral vectors, polyethyleneglycol, or other methods known to those skilled in the art can be used.

Cells are preferably autologous cells, obtained by biopsy and expandedin culture, although cells from close relatives or other donors of thesame species may be used with appropriate immunosuppression.Immunologically inert cells, such as embryonic or fetal cells, stemcells, and cells genetically engineered to avoid the need forimmunosuppression can also be used. Methods and drugs forimmunosuppression are known to those skilled in the art oftransplantation. A preferred compound is cyclosporin using therecommended dosages.

In the preferred embodiment, cells are obtained by biopsy and expandedin culture for subsequent implantation. Cells can be easily obtainedthrough a biopsy anywhere in the body, for example, skeletal musclebiopsies can be obtained easily from the arm, forearm, or lowerextremities, and smooth muscle can be obtained from the area adjacent tothe subcutaneous tissue throughout the body. To obtain either type ofmuscle, the area to be biopsied can be locally anesthetized with a smallamount of lidocaine injected subcutaneously. Alternatively, a smallpatch of lidocaine jelly can be applied over the area to be biopsied andleft in place for a period of 5 to 20 minutes, prior to obtaining biopsyspecimen. The biopsy can be effortlessly obtained with the use of abiopsy needle, a rapid action needle which makes the procedure extremelysimple and almost painless. With the addition of the anesthetic agent,the procedure would be entirely painless. This small biopsy core ofeither skeletal or smooth muscle can then be transferred to mediaconsisting of phosphate buffered saline. The biopsy specimen is thentransferred to the lab where the muscle can be grown utilizing theexplant technique, wherein the muscle is divided into very pieces whichare adhered to culture plate, and serum containing media is added.Alternatively, the muscle biopsy can be enzymatically digested withagents such as trypsin and the cells dispersed in a culture plate withany of the routinely used medias. After cell expansion within theculture plate, the cells can be easily passaged utilizing the usualtechnique until an adequate number of cells is achieved.

III. Methods for Implantation

Unlike other prior art methods for making implantable matrices, thepresent method uses the recipient or an animal as the initial bioreactorto form a fibrous tissue-polymeric construct which optionally can beseeded with other cells and implanted. The matrix becomes infiltratedwith fibrous tissue and/or blood vessels over a period ranging frombetween one day and a few weeks, most preferably one and two weeks. Thematrix is then removed and implanted at the site where it is needed.

In one embodiment, the matrix is formed of polymer fibers having aparticular desired shape, that is implanted subcutaneously. The implantis retrieved surgically, then one or more defined cell types distributedonto and into the fibers. In a second embodiment, the matrix is seededwith cells of a defined type, implanted until fibrous tissue has growninto the matrix, then the matrix removed, optionally cultured further invitro, then reimplanted at a desired site.

The resulting structures are dictated by the matrix construction,including architecture, porosity (% void volume and pore diameter),polymer nature including composition, crystallinity, molecular weight,and degradability, hydrophobicity, and the inclusion of otherbiologically active molecules.

This methodology is particularly well suited for the construction ofvalves and tubular structures. Examples of valves are heart valves andvalves of the type used for ventricular shunts for treatment ofhydrocephaly. A similar structure could be used for an ascites shunt inthe abdomen where needed due to liver disease or in the case of alymphatic obstructive disease. Examples of tubular structures includeblood vessels, intestine, ureters, and fallopian tubes.

The structures are formed at a site other than where they are ultimatelyrequired. This is particularly important in the case of tubularstructures and valves, where integrity to fluid is essential, and wherethe structure is subjected to repeated stress and strain.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLE 1

Tissue Engineering of Heart Valves

Valvular heart disease is a significant cause of morbidity andmortality. Construction of a tissue engineered valve using livingautologous cells offers advantages over currently used mechanical orglutaraldehyde fixed xenograft valves.

Methods and Materials

A tissue engineered valve was constructed by seeding a syntheticpolyglycolic acid (PGA) fiber based matrix with dissociated fibroblastsand endothelial cells harvested from a donor sheep heart valve. Thecells were grown to confluence and split several times to increase thecell number. A mixed cell population including myofibroblasts andendothelial cells was obtained. The endothelial cells were labeled withan Ac-Dil-LDL fluorescent antibody obtained from a commercial source andsorted in a cell-sorting machine to yield a nearly pure endothelial cellpopulation (LDL+) and a mixed cell population containing myofibroblastsand endothelial cells (LDL−). A PGA mesh (density 76.9 mg/ml andthickness 0.68 mm) was seeded with the mixed cell population and grownin culture. When the myofibroblasts reached confluence, endothelialcells were seeded onto the surface of the fibroblast/mesh constructs andgrown into a single monolayer.

Immunohistochemical evaluation of constructs with antibodies againstfactor VIII, a specific marker for endothelial cells, revealed thattissue engineered valves histologically resemble native valve tissue.The effects of physiological flow on elastin and collagen productionwithin the ECM were examined in a bioreactor and implanted in a sheep todetermine if the constructs had the required pliability and mechanicalstrength for use in patients.

EXAMPLE 2

Tissue Engineering of Vascular Structures

Vascular smooth muscle tubular structures using a biodegradablepolyglycolic acid polymer scaffold have been developed. The techniqueinvolves the isolation and culture of vascular smooth muscle cells, thereconstruction of a vascular wall using biodegradable polymer, andformation of the neo-tissue tubes in vitro. The feasibility ofengineering vascular structures by coculturing endothelial cells withfibroblasts and smooth muscle cells on a synthetic biodegradable matrixin order to create tubular constructs which histologically resemblenative vascular structures was also demonstrated.

Methods

In a first set of studies, bovine and ovine endothelial cells, smoothmuscle cells, and fibroblasts were isolated using a combination ofstandard techniques including collagenase digestion and explantation.These cells were then expanded in tissue culture. All cells were grownin Delbecco's modified Eagle's media supplemented with 10% fetal bovineserum, 1 % antibiotic solution, and basic fibroblast growth factor.Mixed colonies were purified using dilutional cloning. Thirty (N=30) twoby two centimeter polyglycolic acid (PGA) fiber meshes (thickness=0.68mm, density=76.9 mg/cc) were then serially seeded with 5×10⁵ fibroblastsand smooth muscle cells and placed in culture. Five (N=5) 85% PGA, 15%polylatic acid tubular constructs (length=2 cm, diameter=0.8 cm) wereseeded in a similar fashion. After the fibroblasts and smooth musclecell constructs had grown to confluence (mean time 3 weeks), 1×10⁶endothelial cells were seeded onto them and they were placed in culturefor one week. These vascular constructs were then fixed in a paraffin,sectioned and analyzed using immunohistochemical staining for factorVIII (specific for endothelial cells) and desmin (specific for musclecells).

In a second set of studies, smooth muscle cells were obtained byharvesting the media from the artery of a lamb using standard explanttechniques. Cells were expanded in culture through repeated passages andthen seeded on the biodegradable polymer scaffold at a density of 1×10⁶cells per cm² of polymer. The cell-polymer constructs were formed intotubes with internal diameters ranging from 2 mm to 5 mm and maintainedin vitro for 6 to 8 weeks.

Results

Microscopic examination of all constructs in the first study (N=30/N=5)revealed that both types of constructs had achieved the properhistological architecture and resembled native vessels after one week.Immunohistochemical staining confirmed that endothelially lined smoothmuscle/fibroblast tubes had been created. The extracellular matrices(ECM) of the vascular constructs were examined in order to determine thecomposition of elastin and collagen types I and III, the ECM moleculeswhich determine the physical characteristics of native vascular tissues.

The results of the second study show that vascular smooth muscle tubeswhich retain their structure can be successfully formed using apolyglycolic acid polymer scaffold. The biodegradable polymer wasabsorbed over time, leaving a neo-tissue vascular smooth muscle tube.

EXAMPLE 3

Engineered bone from PGA Polymer Scaffold and Periosteum

The ability to create bone from periosteum and biodegradable polymer mayhave significant utility in reconstructive orthopedic and plasticsurgery. Polyglycolic acid (PGA) is a preferred material for forming abiodegradable matrix which can be configured to a desirable shape andstructure. This study was conducted to determine whether new boneconstructs can be formed from periosteum or periosteal cells placed ontoPGA polymer.

Materials and Methods

Bovine periosteum, harvested from fresh calf limbs, was placed eitherdirectly onto PGA polymer (1×1 cm) or onto tissue culture dishes forperiosteal cell isolation. The periosteum/PGA construct was cultured forone week in MEM 199 culture media with antibiotics and ascorbic acid,then implanted into the dorsal subcutaneous space of nude mice.Periosteal cell, cultured from pieces of periosteum for two weeks, wereisolated into cell suspension and seeded (approximately 1 to 3×10⁷cells) onto PGA polymer (1 ×1 cm); after one week in culture, theperiosteal cell seeded polymer was implanted into the subcutaneous spaceof nude mice. Specimens, harvested at 4, 8, and 14 week intervals, wereevaluated grossly and histologically.

Results

The periosteum/PGA constructs showed an organized cartilage matrix withearly evidence of bone formation at four weeks, a mixture of bone andcartilage at 8 weeks, and a complete bone matrix at 14 weeks. Constructscreated from periosteal cells seeded onto polymer showed presence ofdisorganized cartilage at 4 and 8 weeks, and a mixture of bone andcartilage at 14 weeks. Periosteum placed directly onto polymer will forman organized cartilage and bone matrix earlier than constructs formedfrom periosteal cell seeded polymer. This data indicates that PGA is aneffective scaffold for periosteal cell attachment and migration toproduce bone, which may offer new approaches to reconstructive surgery.

EXAMPLE 4

Bone Reconstruction with Tissue Engineered Vascularized Bone

The aim of this study was to determine if new vascularized bone could beengineered by transplantation of osteoblast around existing vascularpedicle using biodegradable polymers as cell delivery devices, to beused to reconstruct weight bearing bony defects.

Methods

Osteoblast and chondryocytes were isolated from calf periosteum andarticular cartilage, cultured in vitro for three weeks, then seeded ontoa 1×1 cm non-woven polyglycolic acid (PGA) mesh. After maintenance invitro for one week, cell-polymer constructs were wrapped aroundsaphenous vessels, and implanted into athymic rats for 8 weeks. Theimplants showed gross and histological evidence of vascularized bone orcartilage. At this time, bilateral 0.8 cm femoral shaft defect werecreated in the same rat, and fixed in position with a 3 cm craniofacialtitanium miniplate. The new engineered bone/cartilage construct was thentransferred to the femoral defect on its bilateral vascular pedicle. Atotal of 30 femoral defects were repaired in three groups of animals(each group composed of five animals with defects). Animals in Group 1received implants composed of vascularized bone constructs, animals inGroup 2 with vascular cartilage constructs, and Group 3 animals withblank polymer only.

At six months after surgery, the animals were studied radiographicallyfor evidence of new bone formation at the site of the defect. Euthanasiawas then performed by anesthetic overdose and each experimented femurwas removed. Gross appearance was recorded and histological studiesperformed using hematoxylin and eosin (H & E) staining.

Results

Group 1 defect showed evidence of new bone formation around the defect.Neither Group 2 nor Group 3 defect showed any radiographic evidence ofhealing or bone formation. Grossly, Group 1 animals developed exuberantbony callus formation and healing of the defect. The animals in Group 2showed filling of the bony defect with cartilaginous tissue, whereas allof the animals in Group 3 either developed a fibrous non-union or simpleseparation of both bony fragments with soft tissue invasion of thedefect. The histological studies showed new bone formation in all Group1 animals, new cartilage formation in all Group 2 animals, and fibroustissue invasion in all Group 3 animals.

Conclusion

In conclusion, it was possible to engineer vascularized bone andcartilage grafts, which could be used to repair bone defects in the ratfemur. Engineered tissue maintained the characteristics of the tissuesform which the cells were originally isolated.

EXAMPLE 5

Engineering of Composite Bone and Cartilage

The ability to construct a composite structure of bone and cartilageoffers a significant modality in reconstructive plastic and orthopedicsurgery. The following study was conducted to engineer a bone andcartilage composite structure using periosteum, chondrocytes andbiodegradable polymer and to direct bone and cartilage formation byselectively placing periosteum and chondrocytes onto the polymerscaffold.

Methods and materials

Bovine periosteum and cartilage were harvested from newborn calf limbs.Periosteum (1.5×2.0 cm) was wrapped around a polyglycolic acid/polyL-lactic acid co-polymer tube (3 cm in length, 3 mm in diameter),leaving the ends exposed. The cartilage pieces were enzymaticallydigested with collagenase, and chondrocytes (2×10⁷ cells) were seededonto each end of the exposed polymer. The composite construct wascultured for seven days in Medium 199 with antibiotics, fetal bovineserum, and ascorbic acid at 37° C. with 5% CO₂. Eight constructs werethen implanted into the dorsal subcutaneous space of eight nude mice.After 8 to 14 weeks in vivo, the implants were harvested and evaluatedgrossly and histologically.

Results

All implants formed into cylindrical shapes, flattened at the ends. Thecentral portion of the implant formed into a bony matrix and the ends ofthe specimens formed into cartilage, approximately where the periosteumand chondrocytes were placed. Histological sections showed an organizedmatrix of bone and cartilage with a distinct transition between bone andcartilage.

Conclusions

The results show that periosteum and chondrocytes placed onto abiodegradable polymer will form into a composite tissue of bone andcartilage. Moreoever, bone and cartilage composite formation withselective placement of periosteum and chondrocytes on a biodegradablepolymer scaffold was shown.

EXAMPLE 6

Implantation of Matrix for Ingrowth of Fibrous Tissue to IncreaseMechanical Properties and Cell Survival

The following study was conducted to increase the mechanical strengthand pliability of the heart valve leaflets or other engineered tissuessuch as those for use as blood vessels.

Methods

A PGA mesh as described in Example 1 or 2 was implanted subcutaneouslyin an animal, then removed after a period of one to two weeks.Fibroblasts migrated into the polymeric mesh while it was implanted. Theimplant was then seeded with other cells such as chondrocytes orendothelial cells and cultured in vitro for an additional period oftime.

Results

The resulting implant was shown to have greater mechanical strength andpliability than implants formed solely by seeding of dissociated cells.

Modifications and variations of the method and compositions describedherein will be obvious to those skilled in the art from the foregoingdetailed description. Such modifications and variations are intended tocome within the scope of the appended claims.

We claim:
 1. A method for making a cell-matrix construct for use as aheart valve or blood vessel comprising implanting into an animal at afirst site a fibrous matrix formed of a synthetic biodegradable polymerhaving seeded therein a mixture of cells selected from the groupselected from endothelial cells, myofibroblasts, skeletal muscle cells,vascular smooth muscle cells, myocytes, fibromyoblasts, and ectodermalcells, wherein the matrix is formed of a biocompatible, biodegradablepolymer, and implanting into an animal or human the matrix at a sitewhere the resulting cell-construct is needed.
 2. The method of claim 1further comprising seeding the matrix with dissociated parenchymal orconnective tissue cells.
 3. The method of claim 1 wherein the matrix isfirst cultured at a first site in a patient prior to being implanted ata second site.
 4. The method of claim 1 wherein the matrix is a heartvalve and is implanted in the heart.
 5. The method of claim 1 whereinthe cell-matrix construct is seeded with vascular smooth muscle cellsand endothelial cells is implanted to form a valve.
 6. The method ofclaim 5 wherein the valve is a heart valve.
 7. The method of claim 1wherein the cell-matrix construct is seeded with endothelial cells andimplanted to form a blood vessel.